Lesions of the cholinergic basal forebrain (CBF) system in animals using an immunotoxin produce robust behavioral deficits and mimic some aspects of Alzheimer's disease (AD). This model is useful for testing both the role of acetylcholine in cognitive processes and new therapeutic strategies such as gene therapy. The ability to control gene expression is an important requirement of this technology for clinical application. The vector systems being explored in this proposal may be exogenously regulated. This proposal will investigate mechanisms of restoring neurotransmitter function in animals with CBF immunotoxin lesions by 1) enhancing ACh release postgrafting via choline supplementation, and 2) transplanting cells capable of repressing or inducing the transgene for choline acetyltransferase (ChAT). The repressible system (tetracycline), and the inducible system (eccdysteroid), modulates gene expression of ChAT in a rapid, reversible and highly specific fashion. In the first series of experiments, the investigators will determine whether different doses of exogenously administered choline can augment the production and release of acetylcholine from genetically engineered fibroblasts grafted to the cortex and hippocampus of rats following immunotoxic lesions of the CBF. Once an increase in release has been determined, behavioral effects of this augmented release will be determined. For the second and third series of experiments, they will use the regulatable cell lines that either induce or repress ChAT expression after administration of doxycycline or Muristerone A, respectively. In their immunotoxin lesion they will demonstrate that acetylcholine released from these cells after grafting to the neocortex and hippocampus is both necessary and sufficient for behavioral recovery. The effects of these regulatable genes will be tested in a spatial and non-spatial task after determining the dose and duration of the exogenous compound needed to induce or repress gene expression. If successful, the experiments will demonstrate that release of ACh can be exogenously controlled in animals and results in significant behavioral improvement. The control of transmitter release is necessary before grafting or direct gene insertion experiments can be entertained in humans in order to be able to safely terminate delivery of the gene product. These approaches will not only be applicable for AD, but other neurodegenerative disorders and for delivering other transgenes of interest.
